An ultimate goal in the treatment of HIV-infected persons is to prevent disease progression. Therapeutic HIV vaccines have been developed to protect HIV-infected individuals from progression to AIDS. Current HIV vaccines include Remune, an inactivated HIV-1 lacking the gp120 glycoprotein, VaxSyn, a recombinant gp160, and p24 virus-like particles. Another approach in the treatment of HIV-infected persons is to use chemotherapy to reduce viral load, followed by immunotherapy to stimulate desirable HIV-specific immune responses, such as those observed among long-term asymptomatic HIV-infected individuals.
During the past ten years, chemotherapy for HIV-infected individuals has advanced rapidly. Current treatment consists of a cocktail of anti-retroviral drugs termed highly active anti-retroviral therapy (HAART). While HAART reduces viral load, it does not eradicate HIV (Saag and Kilby, Nature Medicine 5(6): 609-11 (1999)). In addition, HAART is often associated with drug-related toxicity and the selection of drug-resistant mutants. Additional strategies would therefore be desirable to treat HIV-infected individuals.
A body of work suggests that continued health maintenance in patients with long-term non-progressive HIV infection can be attributed to effective CTL responses in general, and gag-specific CTL responses in particular, each driven by vigorous antigen-specific CD4+ T helper cells (Gotch et al., Immunol. Rev.: 170173-82 (1999)). Thus, one strategy in the treatment of HIV-infected individuals involves the reduction of viral load with HAART, followed by modulation of the host's immune response such that it mimics the protective immunogenic response found among long-term asymptomatic HIV-infected patients (Gotch et al., Immunol. Rev.: 170173-82 (1999)).
Modulation of the host's immune response can be carried out with the use of a viral vaccine which induces in the host both CTL and CD4+ T helper cell responses. Thus, a viral vaccine which induces HIV-specific T cell responses, and which is further capable of inducing CTL and CD4+ T helper cell responses, would be of great utility in the treatment of HIV-infected individuals.
Picornaviruses are attractive for use as viral vaccine vectors since they induce B and T cell immunity. In addition, the enteroviruses belonging to the Picornaviridae family, which include poliovirus and coxsackievirus, are known to induce mucosal immunity which is thought to be an important line of defense for pathogens (including HIV) that gain access via a mucosal port of entry. Thus, coxsackieviruses in particular should be considered.
Coxsackieviruses are subdivided into two serogroups, A and B, which comprise 24 and 6 serotypes, respectively (Rueckert, R. R., Fundamental Virology. 3rd ed. Philadelphia: Lippincott-Raven, 1996). Coxsackieviruses of the B group have been implicated in diseases such as pancreatitis, type I insulin-dependent diabetes mellitus, myocarditis and myositis. The existence of variants within a single serotype contributes to the variability in the pathogenesis of coxsackievirus infections. An avirulent coxsackievirus would be a suitable candidate for in vivo expression of HIV sequences capable of stimulating both CTL and CD4+ T helper cell responses.
Picornaviruses, which include coxsackievirus, are among the smallest RNA viruses, with a diameter of 30 mm (reviewed in (Metnick, J. L., Fields Virology. In: Fields B N, Knipe D M, Howley P M, et al. editors. Third ed. Philadelphia: Lippincott-Raven Publishers, 1996: 655-705). The coxsackievirus virion consists of a protein shell surrounding an RNA genome. The protein shell contains sixty copies of each of four capsid proteins, VP1, VP2, VP3 and VP4 that form an icosahedron. The enteroviral genome consists of a single-stranded RNA of positive polarity. Excluding the poly(A) tract, the genome-of coxsackievirus B4 consists of 7,395 nucleotides and is composed of a 5′ untranslated (UTR) region of 743 nucleotides, a 3′ UTR of 105 nucleotides and an open reading frame encoding a polyprotein of 2,183 amino acids which undergoes multiple cleavages (Jenkins et al., J. Gen. Virol. 68: 1835-1848 (1987)). The open reading frame is divided into three regions, P1, P2 and P3. The four capsid proteins, VP1 through VP4, are encoded within the P1 region while the non-structural proteins that are involved in viral replication are encoded within the P2 and P3 regions. Two B cell epitopes within the VP1 coat protein of CB4-V have recently been identified (Halim and Ramsingh, Virol. 269: 86-94 (1999)). A linear B cell epitope spans residues 68 to 82 that corresponds to parts of beta strand B and the BC-loop. A conformational epitope, analogous to antigenic site 1B of poliovirus, is predicted to contain sequences from both the DE- and BC-loops of VP1.
Efforts to exploit the picornaviruses as expression vectors have focused mainly on poliovirus. Several strategies have been used to express a variety of sequences within poliovirus vectors. Small antigenic epitopes have been expressed within the BC-loop of the VP1 capsid protein (Dedieu et al., J. Virot. 66: 3161-3167 (1992); Evans et al., Nature 339: 385-388 (1989); Jenkins et al., J. Virot. 64: 1201-1206 (1990); Murdin et al., Infect. Immun. 61: 4406-4414 (1993)). The resulting chimeras were either genetically stable, genetically unstable or non-viable. The stability of the recombinants seemed to depend on the retention of some flanking viral loop sequences and the size of the inserted sequence.
Two approaches have been used to generate live poliovirus vectors. One approach expresses foreign sequences in dicistronic vectors containing an additional internal ribosome entry site (IRES) (Lu et al., J. Virol. 69: 4797-4806 (1995)). While the dicistronic vector system resulted in replication-competent viruses, they were genetically unstable after just a few passages in tissue culture. Another strategy positions foreign sequence, flanked by artificial protease cleavage sites, at different sites within the polyprotein (Andino et al., J. Virot. 72: 20-31 (1994)). A larger than normal precursor polyprotein is synthesized and subsequently cleaved into its normal products and the foreign protein. Lately, some controversy has arisen regarding the utility of this strategy. Recombinants constructed by fusing the open reading frame (ORF) of the green fluorescent protein gene or the gag gene to the N-terminus of the poliovirus polyprotein were severely impaired in viral replication and genetically unstable (13). It is known that the site of insertion of the foreign sequence influenced viral replication (Tang et al., J. Virot. 71: 7841-7850 (1997)). A small amino-terminus insertion delayed virus maturation and yielded a thermosensitive particle. However, insertion at the junction between the P1 and P2 regions yielded a chimeric poliovirus that replicated like the wild type virus. While genetic instability remained a problem, the situation could be partially alleviated by altering the sequences flanking the insertion point. However, since poliovirus has been targeted for global eradication, the feasibility of using live poliovirus as a vector becomes uncertain.
Recent studies have led to the identification of an attenuated-live coxsackievirus variant, CB4-P (Ramsingh et al., Virus Res. 14: 347-58 (1989); Caggana et al., J. Virol. 67: 4797-803 (1993); Ramsingh et al., J. Virol. 71(11): 8690-7 (1997)). The major determinant of virulence was mapped to the 5′ end of the viral genome through the use of recombinant chimeric viruses derived from cDNA clones of the CB4-P variant and a virulent virus. The 5′ end of the genome encompasses both the 5′ untranslated region (UTR) and the P1 region, which encodes the four capsid proteins (Ramsingh et al., Virus Res. 23: 281-92 (1992)). Comparison of sequence data in the 5′ region between the CB4-P variant and a virulent pancreatropic variant of coxsackievirus, CB4-V, revealed five mutations in the CB4-P variant that resulted in amino acid substitutions in VP1, VP2, and VP4. In particular, one residue, Thr-129 of VP1, is a major determinant of virulence in the 5′UTR for coxsackievirus B4 (Caggana et al., J. Virol. 67: 4797-803 (1993)). An arginine residue at position 16 of VP4 also influences virulence but to a lesser extent than thr-129 of VP1 (Ramsingh and Collins, J. Virol. 69: 7278-7281 (1995)). The potential of this variant for use as a viral vector for delivery of heterologous polypeptides to a host has yet to be explored.
At least one attenuated strain of coxsackievirus has been used experimentally as a recombinant viral vaccine. Group B coxsackieviruses (CVB) cause human myocarditis, in which human adenovirus type 2 (Ad2) has been implicated as an agent. It has recently been demonstrated that an attenuated group B coxsackieviruses type 3 (CVB3) vector can stably express an antigenic polypeptide of Ad2 from within the vector open reading frame to ultimately elicit a protective immune response against both viruses in mice (Hofling et al., J. Virol. 74: 4570-8 (2000)).